DOC2B Acts as a Calcium Switch and Enhances Vesicle Fusion

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.

[1]  B. Davletov,et al.  Real-time assay for monitoring membrane association of lipid-binding domains , 2008, Analytical biochemistry.

[2]  C. Fernández-Peruchena,et al.  Myosin II Contributes to Fusion Pore Expansion during Exocytosis* , 2008, Journal of Biological Chemistry.

[3]  A. Mezer,et al.  Vesicle Priming and Recruitment by ubMunc13-2 Are Differentially Regulated by Calcium and Calmodulin , 2008, The Journal of Neuroscience.

[4]  V. Shahin,et al.  Synaptotagmin perturbs the structure of phospholipid bilayers. , 2008, Biochemistry.

[5]  D. Atlas,et al.  Depolarization-Evoked Secretion Requires Two Vicinal Transmembrane Cysteines of Syntaxin 1A , 2007, PloS one.

[6]  D. Bruns,et al.  v-SNARE Actions during Ca2+-Triggered Exocytosis , 2007, Cell.

[7]  O. Yizhar,et al.  Multiple functional domains are involved in tomosyn regulation of exocytosis , 2007, Journal of neurochemistry.

[8]  G. Martin,et al.  Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets , 2007, The Journal of cell biology.

[9]  B. Ke,et al.  Doc2β Is a Novel Munc18c-interacting Partner and Positive Effector of Syntaxin 4-mediated Exocytosis* , 2007, Journal of Biological Chemistry.

[10]  A. Neiman,et al.  Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage* , 2007, Journal of Biological Chemistry.

[11]  B. Davletov,et al.  Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells. , 2007, Cell calcium.

[12]  M. Kozlov,et al.  How Synaptotagmin Promotes Membrane Fusion , 2007, Science.

[13]  Ute Becherer,et al.  Primed Vesicles Can Be Distinguished from Docked Vesicles by Analyzing Their Mobility , 2007, The Journal of Neuroscience.

[14]  A. Neiman,et al.  1 PLD 1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granule at a late stage , 2007 .

[15]  M. Verhage,et al.  Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells , 2006, Neuroscience.

[16]  E. Neher,et al.  A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse , 2006, Pflügers Archiv.

[17]  Y. Henis,et al.  FRAP beam-size analysis to measure palmitoylation-dependent membrane association dynamics and microdomain partitioning of Ras proteins. , 2006, Methods.

[18]  Corey Smith,et al.  Physiological stimulation regulates the exocytic mode through calcium activation of protein kinase C in mouse chromaffin cells. , 2006, The Biochemical journal.

[19]  Reinhard Jahn,et al.  SNAREs — engines for membrane fusion , 2006, Nature Reviews Molecular Cell Biology.

[20]  M. Verhage,et al.  Dissecting docking and tethering of secretory vesicles at the target membrane , 2006, The EMBO journal.

[21]  Michael Dybbs,et al.  Antagonistic Regulation of Synaptic Vesicle Priming by Tomosyn and UNC-13 , 2006, Neuron.

[22]  U. Becherer,et al.  Vesicle pools, docking, priming, and release , 2006, Cell and Tissue Research.

[23]  M. Verhage,et al.  DOC2A and DOC2B are sensors for neuronal activity with unique calcium‐dependent and kinetic properties , 2006, Journal of neurochemistry.

[24]  M. Spira,et al.  Calcium concentration threshold and translocation kinetics of EGFP-DOC2B expressed in cultured Aplysia neurons. , 2006, Cell calcium.

[25]  U. Matti,et al.  Identification of the Minimal Protein Domain Required for Priming Activity of Munc13-1 , 2005, Current Biology.

[26]  D. Sulzer,et al.  Analysis of exocytotic events recorded by amperometry , 2005, Nature Methods.

[27]  Corey Smith,et al.  Activity-Dependent Differential Transmitter Release in Mouse Adrenal Chromaffin Cells , 2005, The Journal of Neuroscience.

[28]  M. Verhage,et al.  Two distinct genes drive expression of seven tomosyn isoforms in the mammalian brain, sharing a conserved structure with a unique variable domain , 2005, Journal of neurochemistry.

[29]  Tim J. Craig,et al.  Regulation of the Fusion Pore Conductance during Exocytosis by Cyclin-dependent Kinase 5* , 2004, Journal of Biological Chemistry.

[30]  J. Hancock,et al.  Three Separable Domains Regulate GTP-Dependent Association of H-ras with the Plasma Membrane , 2004, Molecular and Cellular Biology.

[31]  D. Bruns,et al.  Detection of transmitter release with carbon fiber electrodes. , 2004, Methods.

[32]  J. Barclay,et al.  Syntaxin/Munc18 Interactions in the Late Events during Vesicle Fusion and Release in Exocytosis*[boxs] , 2004, Journal of Biological Chemistry.

[33]  J. Jaiswal,et al.  Synaptotagmin VII Restricts Fusion Pore Expansion during Lysosomal Exocytosis , 2004, PLoS biology.

[34]  M. Verhage,et al.  Ca2+-induced Recruitment of the Secretory Vesicle Protein DOC2B to the Target Membrane*[boxs] , 2004, Journal of Biological Chemistry.

[35]  M. Jackson,et al.  Transmembrane Segments of Syntaxin Line the Fusion Pore of Ca2+-Triggered Exocytosis , 2004, Science.

[36]  B. Davletov,et al.  Synaptotagmin Interaction with the Syntaxin/SNAP-25 Dimer Is Mediated by an Evolutionarily Conserved Motif and Is Sensitive to Inositol Hexakisphosphate* , 2004, Journal of Biological Chemistry.

[37]  M. Jackson,et al.  Fusion Pore Dynamics Are Regulated by Synaptotagmin•t-SNARE Interactions , 2004, Neuron.

[38]  J. B. Sørensen.  Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles , 2004, Pflügers Archiv.

[39]  Colin Rickman,et al.  High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E* , 2004, Journal of Biological Chemistry.

[40]  T. Sudhof,et al.  The synaptic vesicle cycle. , 2004, Annual review of neuroscience.

[41]  T. Südhof,et al.  Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2+ , 2003, The Journal of general physiology.

[42]  P. De Camilli,et al.  Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  E. Neher,et al.  Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser187 Potentiates Vesicle Recruitment , 2002, The Journal of Neuroscience.

[44]  E. Neher,et al.  Emerging Roles of Presynaptic Proteins in Ca++-Triggered Exocytosis , 2002, Science.

[45]  K. Broadie,et al.  The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans , 2002, Current Opinion in Neurobiology.

[46]  H. McMahon,et al.  Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[47]  A. Morales,et al.  Nongenomic regulation of the kinetics of exocytosis by estrogens. , 2002, The Journal of pharmacology and experimental therapeutics.

[48]  J. Barclay,et al.  Splitting the quantum: regulation of quantal release during vesicle fusion , 2002, Trends in Neurosciences.

[49]  M. Jackson,et al.  Synaptotagmin Modulation of Fusion Pore Kinetics in Regulated Exocytosis of Dense-Core Vesicles , 2001, Science.

[50]  H. Palfrey,et al.  Quantal Size Is Dependent on Stimulation Frequency and Calcium Entry in Calf Chromaffin Cells , 2001, Neuron.

[51]  E. Jorgensen,et al.  An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming , 2001, Nature.

[52]  S. Chasserot-Golaz,et al.  Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells , 2001, The EMBO journal.

[53]  D. Bruns,et al.  SNAREs are concentrated in cholesterol‐dependent clusters that define docking and fusion sites for exocytosis , 2001, The EMBO journal.

[54]  T. Voets.  Dissection of Three Ca2+-Dependent Steps Leading to Secretion in Chromaffin Cells from Mouse Adrenal Slices , 2000, Neuron.

[55]  Ralf Schneggenburger,et al.  Intracellular calcium dependence of transmitter release rates at a fast central synapse , 2000, Nature.

[56]  M. Gollasch,et al.  Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i , 2000 .

[57]  E. Neher,et al.  Munc13‐1 acts as a priming factor for large dense‐core vesicles in bovine chromaffin cells , 2000, The EMBO journal.

[58]  M. Gollasch,et al.  Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i). , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[59]  S. Itohara,et al.  Doc2α is an activity‐dependent modulator of excitatory synaptic transmission , 1999, The European journal of neuroscience.

[60]  A. Brunger,et al.  Crystal Structure of the Cytosolic C2a-C2b Domains of Synaptotagmin III , 1999, The Journal of cell biology.

[61]  G. Augustine,et al.  Proteins involved in synaptic vesicle trafficking , 1999, The Journal of physiology.

[62]  R. Chow,et al.  Transient, Phorbol Ester-induced DOC2-Munc13 Interactions in Vivo * , 1999, The Journal of Biological Chemistry.

[63]  Y. Takai,et al.  Presynaptic Mechanism for Phorbol Ester-Induced Synaptic Potentiation , 1999, The Journal of Neuroscience.

[64]  U. Ashery,et al.  An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system. , 1999, European journal of cell biology.

[65]  E. Neher,et al.  Early requirement for α‐SNAP and NSF in the secretory cascade in chromaffin cells , 1999 .

[66]  E. Neher,et al.  Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells. , 1999, The EMBO journal.

[67]  Tobias Meyer,et al.  Protein Kinase C as a Molecular Machine for Decoding Calcium and Diacylglycerol Signals , 1998, Cell.

[68]  Y. Takai,et al.  Role of the Doc2α–Munc13–1 interaction in the neurotransmitter release process , 1998 .

[69]  T. Martin,et al.  Docking and fusion in neurosecretion. , 1998, Current opinion in cell biology.

[70]  Leonard K. Kaczmarek,et al.  High-frequency firing helps replenish the readily releasable pool of synaptic vesicles , 1998, Nature.

[71]  T. Südhof,et al.  Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2‐domain? , 1998, The EMBO journal.

[72]  Tao Xu,et al.  Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity , 1998, Nature Neuroscience.

[73]  E. Neher,et al.  Cytosolic Ca2+ Acts by Two Separate Pathways to Modulate the Supply of Release-Competent Vesicles in Chromaffin Cells , 1998, Neuron.

[74]  T. Sasaki,et al.  Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[75]  R. Wightman,et al.  Spatio-temporal resolution of exocytosis from individual cells. , 1998, Annual review of biophysics and biomolecular structure.

[76]  G. Alvarez de Toledo,et al.  The exocytotic event in chromaffin cells revealed by patch amperometry , 1997, Nature.

[77]  Takuya Sasaki,et al.  Physical and Functional Interactions of Doc2 and Munc13 in Ca2+-dependent Exocytotic Machinery* , 1997, The Journal of Biological Chemistry.

[78]  R. Burgoyne,et al.  Common mechanisms for regulated exocytosis in the chromaffin cell and the synapse. , 1997, Seminars in cell & developmental biology.

[79]  T. Südhof,et al.  DOC2 Proteins in Rat Brain: Complementary Distribution and Proposed Function as Vesicular Adapter Proteins in Early Stages of Secretion , 1997, Neuron.

[80]  Takuya Sasaki,et al.  Doc2 Enhances Ca-dependent Exocytosis from PC12 Cells (*) , 1996, The Journal of Biological Chemistry.

[81]  R. Wightman,et al.  Temporally resolved, independent stages of individual exocytotic secretion events. , 1996, Biophysical journal.

[82]  R. Burgoyne,et al.  Ca2+ and secretory-vesicle dynamics , 1995, Trends in Neurosciences.

[83]  T. Sasaki,et al.  Doc2: a novel brain protein having two repeated C2-like domains. , 1995, Biochemical and biophysical research communications.

[84]  E. Neher,et al.  A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. , 1993, Science.

[85]  R. Holz,et al.  Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components. , 1992, The Journal of biological chemistry.

[86]  B. Katz,et al.  The role of calcium in neuromuscular facilitation , 1968, The Journal of physiology.